Vincent S. Gallicchio

Didox (A Novel Ribonucleotide Reductase Inhibitor) Overcomes bcl-2 Mediated Radiation Resistance in Prostate Cancer Cell Line PC-3

In this study, we investigated the influence of bcl-2 overexpression on the radiosensitizing potential of Didox (DX; 3,4-Dihydroxybenzohydroxamic acid), a novel ribonucleotide reductase inhibitor, in p53-null prostate cancer cell line PC-3. The PC-3 cells were transfected with vector alone or ectopically overexpressed with CMV-bcl-2 construct. The effect of radiation (IR) or DX alone and in combination (pre and post IR exposure of DX) on cell survival was determined by colony-forming assay. The impact of these two treatments on the cell cycle was determined by flow cytometry. To further understand the molecular mechanism of DX-mediated radiosensitization, induction of pro-survival and pro-apoptotic factors were determined by Western blot and gel-shift assays respectively. When compared to PC-3/bcl-2 cells (SF2=0.84; D0=437cGy), the PC-3/vector cells (SF2=0.4; D0=235cGy) were significantly sensitive to ionizing radiation (p

Farnesyltransferase inhibitor (L-744,832) restores TGF-β type II receptor expression and enhances radiation sensitivity in K-ras mutant pancreatic cancer cell line MIA PaCa-2

Activated ras is known to dysregulate TGF-β signaling by altering the expression of TGF-β type II receptor (RII). It is well documented that tumor cells harboring mutant ras are more resistant to radiation than cells with wild-type ras. In this study, we hypothesized that the use of farnesyltransferase inhibitor (FTI, L-744,832) may directly restore TGF-β signaling through RII expression via ras dependent or independent pathway leading to induction of radiation sensitivity. Two pancreatic cancer cell lines, BxPC-3 and MIA PaCa-2 were used in this study. FTI inhibited farnesylation of Ras protein more significantly in MIA PaCa-2 than BxPC-3 cells. In contrast, MIA PaCa-2 cells were resistant to radiation when compared to BxPC-3 cells. BxPC-3 cells were more resistant to FTI than MIA PaCa-2 cells. In combination treatment, no significant radiosensitizing effect of FTI was observed in BxPC-3 cells at 5 or 10 μM. However, in MIA PaCa-2 cells, a significant radiosensitizing effect was observed at both 5 and 10 μM concentrations (P>0.004). The TGF-β effector gene p21waf1/cip1 was elevated in combination treatment in MIA PaCa-2 but not in BxPC-3 cells. In MIA PaCa-2 cells, FTI induced TGF-β responsive promoter activity as assessed by 3TP-luciferase activity. A further induction of luciferase activity was observed in MIA PaCa-2 cells treated with radiation and FTI. Induction of TGF-β signaling by FTI was mediated through restoration of the RII expression, as demonstrated by RT–PCR analysis. In addition, re-expression of RII by FTI was associated with a decrease in DNA methyltransferase 1 (DNMT1) levels. Thus, these findings suggest that the L-744,832 treatment restores the RII expression through inhibition of DNMT1 levels causing induction of TGF-β signaling by radiation and this forms a novel molecular mechanism of radiosensitization by FTI.

Specificity of lithium (li+) to enhance the production of colony stimulating factor (gm-csf) from mitogen-stimulated lymphocytes in vitro.

We report here studies demonstrating the ability of Li+ to increase GM-CSF production from both mitogen-induced spleen and thymus cells prepared as serum-free conditioned media (SF-SCCM, SF-TCCM). GM-CSF activity was both a mitogen and Li+ specific mediated event (P less than 0.001-0.001). Identical cultures prepared with either Na, K, Ca, or Mg did not induce GM-CSF activity as compared to Li. No GM-CSF activity was observed in the absence of mitogen. Furthermore, indomethacin (10(-6) M), a potent inhibitor of prostaglandin (PG) synthesis, produced an even greater enhancement in GM-CSF than control cultures prepared without indomethacin. These data indicate Li may enhance GM-CSF production by inhibiting the ability of PG to decrease GM-CSF production. CFU-Mk colony formation was not significantly influenced by any specific cation-induced mitogen (CM), suggesting Lis ability to stimulate megakaryocytopoiesis may be mediated via a more direct stem cell effect. Furthermore, Li-derived (CM) significantly reduced both CFU-E and BFU-E, while those CMs prepared in the presence of K and Ca significantly increased erythroid colony formation. These effects could be mediated via alterations in the production of BPA. These studies demonstrate the unique capacity of cations to influence the differentiation of committed hematopoietic stem cells possibly by modulating the production of such factors required for hematopoietic differentiation.

Effect of lithium in murine immunodeficiency virus infected animals.

Murine AIDS (MAIDS) is a disease that shows many similarities to human HIV infection. The etiological agent of MAIDS is a defective murine leukemia virus that seems to be able to induce disease in the absence of viral replication. This animal model has been useful in stimulating the search of answers to questions and the formation of new hypotheses related to human AIDS. The monovalent cation lithium can influence a number of immunohematopoietic cell types and cellular processes where proliferation and differentiation occur. We describe here the result of in vivo studies investigating the effect of lithium treatment on MAIDS-infected mice. Viral control and lithium-treated animals were monitored for survival and development of MAIDS pathology. MAIDS animals treated with lithium demonstrated a marked reduction in their development of lymphadenopathy and splenomegaly. Both MAIDS control and lithium-treated virus-infected mice developed evidence of lymphoma; however, the involvement was much more massive both at the gross and microscopic levels in the MAIDS control compared with the lithium-treated mice. These data suggest that lithium may be effective in modulating murine immunodeficiency virus infection and raise important questions related to the potential role lithium may play in the pathophysiological processes associated with retroviral infections.

Analysis of granulocyte-macrophage progenitor cells in patients treated with recombinant interferon alpha-2.

The most common dose-limiting toxicity of alpha interferon has been leukopenia. It is well documented that interferon inhibits human multipotential, erythroid, and granulocyte-macrophage progenitor cells in vitro. Granulocyte-macrophage progenitor cells were evaluated by a colony-forming assay in patients treated with recombinant interferon alpha-2, and results of the assay were correlated with histologic findings in the same bone marrow aspirates and biopsy specimens from the same patients. Eleven patients received 10 million units/m2 interferon subcutaneously thrice weekly for three months. The bone marrow was evaluated on Day-3 (pretreatment) and Day 10 of treatment. Colony-forming granulocyte-macrophage cell count fell from 40.9 +/- 7.6 colonies per 10(5) cells before treatment to 9.3 +/- 1.2 at Day 10 (mean +/- SE, p less than 0.001). It is concluded that the leukopenia induced by interferon is caused by inhibition of maturation of marrow progenitor cells preventing the repopulation of the peripheral blood.

ABILITY OF LITHIUM TO ACCELERATE THE RECOVERY OF GRANULOPOIESIS AFTER SUBACUTE RADIATION INJURY

Lithium stimulates granulopoietic recovery after mice are exposed to 2 Gy. By examining the hematopoietic inductive microenvironment (HIM) using the stromal colony assay, we demonstrate here that lithium, during the two weeks after irradiation, produced less stromal colony suppression than was observed from the irradiated controls. Recovery peaked by day 19 and returned to normal by day 28. This response was also observed in splenic derived stroma. Furthermore, stroma from lithium-irradiated animals supported the in vitro growth of granulocyte-macrophage colonies (CFU-GM) greater than observed from irradiated controls. These data suggest lithium accelerates granulopoietic recovery by first providing for a completely reconstituted and functional HIM.

Lithium stimulation of in vitro granulopoiesis: evidence for mediation via sodium transport pathways

Summary. Lithium (Li) stimulates granulopoiesis both in vitro and in vivo by increasing the number of committed granulocyte‐macrophage colony forming stem cells (CFU‐GM) either through direct or indirect mechanisms. In this report are described further studies designed to investigate if this Li stimulation could be altered by modulating Li transport using agents known to influence monovalent cation transport. Ouabain, a Na/K ATPase inhibitor, added to nonadherent bone marrow cells either before or immediately after the addition of ultra‐pure Li (1 mEq) produced an irreversible reduction in CFU‐GM indicatingthe importance of the Na/K ATPase in these processes. Further studies using the sodium and potassium ionophores gramicidin and valinomycin (5 μg/ml) followed by a delay in the addition of Li (0, 5, 15, 20, 30, 60 and 120 min) reduced CFU‐GM; however, this reduction was less severe in the presence of gramicidin, indicating sodium transport pathways may be required for Li action. To further explore this hypothesis, studies were performed using the more specific sodium transport inhibitors, amiloride and phloretin. Both were effective in reducing the ability of Li to increase CFU‐GM. Studies incorporating the calcium ionophore, A23187, demonstrated that in the presence of Li, a further reduction in CFU‐GM was observed indicating that Li was unable to reverse this A23187 induced reduction in CFU‐GM. These data suggest that in the presence of active calcium transport, the ability of Li to increase CFU‐GM is restricted.

Cation influences on in vitro growth of erythroid stem cells (CFU-e and BFU-e)

SummaryRecent developments have focused on long-term bone marrow cultures and serum-free media to establish clonal hematopoiesis in vitro. Prior investigations have suggested an important role for such ingredients as transferrin, albumin, lipid, selenite, and potassium provided by serum. These factors, when present, can support clonal growth even in the absence of serum. Results reported here further define the importance of the cations K+ and Li+ in the regulation of in vitro erythropoiesis in the presence of both normal and dialyzed fetal calf serum and ouabain. While K+ proved essential for optimal erythroid colony formation, the presence of Li+ in such cultures reduced optimal colony formation of erythroid stem cells. This evidence suggests that in long-term marrow cultures or serum-free media K+ is essential for successful maintenance of self-renewal of erythroid stem cells and their differentiation, and that the monitoring of K+ levels may prove useful in such cultures. Since in vitro erythropoiesis was reduced in the presence of Li+, the mechanisms controlling differentiation of hematopoietic stem cell may be interpreted to be subject to cation influence.

Lithium-stimulated Recovery of Granulopoiesis after Sublethal Irradiation Is Not Mediated Via Increased Levels of Colony Stimulating Factor (CSF)

We have previously demonstrated that lithium (Li) is an effective agent in accelerating the recovery of granulopoiesis following sublethal (2 Gy) whole body irradiation. In this report, studies are described that further define this Li-mediated recovery by measuring the levels of colony-stimulating factor (CSF) present in serum from mice administered 105 micrograms/mouse (total dose) of ultra-pure Li2CO3 for 3 days immediately following irradiation. On days 1-28 following the last lithium dose, the serum was tested for its CSF activity against both normal non-adherent derived bone marrow target cells and non-adherent marrow cells from mice administered cyclophosphamide (200 mg/kg body weight). Serum was assayed at 0.01, 0.1, 1 and 10 per cent final concentration. No significant difference in the total number of CFU-GM was observed from normal marrow using either serum from irradiated mice or lithium-treated and irradiated mice, although the irradiation did produce a 300 per cent rise in CFU-GM colonies compared to normal serum (days 4 and 10-15). From regenerating marrow, we observed a significant difference (P less than or equal to 0.01) in CFU-GM cultured with serum at 0.1 per cent concentration from irradiated and lithium-treated mice compared to irradiated mice without lithium. The presence of CSF was confirmed by its reduced activity in the presence of anti-(CSF). These results suggest (Li) may increase the sensitivity of CFU-GM to CSF, thereby producing more CFU-GM ultimately providing more circulating granulocytes.

Synergistic Action of Recombinant-Derived Murine Interleukin-1 on the Augmentation of Colony Stimulating Activity on Murine Granulocyte-Macrophage Hematopoietic Stem Cells in vitro

This communication reports studies that were designed to investigate the role of recombinant-derived murine interleukin-1 on granulocyte macrophage (CFU-GM) progenitor stem cells in vitro. Interleukin-1 (IL-1, 5-25 units/ml culture) in a dose-dependent fashion, stimulated CFU-GM with a maximum effect at 25 units (147.3 +/- 6.4 colonies/10(5) nonadherent-derived marrow cells). At a higher concentration (50 units/ml) this stimulation was significantly reduced. IL-1, in combination with various types of conditioned media: pokeweed mitogen stimulated spleen cell, mouse lung and WEHI-3 cell, as a source of colony-stimulating activity (CSA), produced more CFU-GM when compared to controls (conditioned media alone). Furthermore, when assayed for their esterase activity, IL-1 increased both types of nonspecific and specific esterase content in CFU-GM colonies. Also, the actual ratio between neutrophilic and monocyte/macrophage colonies was reduced when compared to cultures stimulated in the presence of CSA, indicating that IL-1 increased myeloid differentiation. In the presence of indomethacin, an effective inhibitor of prostaglandin synthesis (1 microgram/ml), greater numbers of CFU-GM were present with IL-1 and/or CSA than without indomethacin. Cultures with anti-CSA demonstrated a reduced number of CFU-GM when plated in the presence of CSA but not with IL-1, demonstrating the specificity of IL-1 to stimulate CFU-GM in the presence of anti-CSA antibody. These results indicate a role for IL-1 in modulating granulopoiesis in vitro.